Recombinant protein production using E. coli containing temperature-sensitive constructs

The principles of large scale process design for the use of E. coli strains containing temperature sensitive constructs were developed. Two strains were characterised in a 14L fermenter, one containing a constant copy number plasmid (pCQV2), and the other an amplifiable copy number plasmid (pMG169). Batch fermentations were performed under kinetic limitation to determine key parameters such as optimum conditions for growth and production, oxygen requirements, and the effects of different lengths of time for the implementation of the temperature shift. These results suggested that on the large scale oxygen limitation was likely to occur. Therefore fermentations were performed with a limiting oxygen transfer rate typical of a 100m3 reactor (50-60 mM.L-1h-1. Batch productivity under oxygen limitation fell by between 84% and 87%. The negative effects of oxygen limitation were removed by fermenter operation under glucose limitation, which yielded maximum batch productivity of the three strategies. At all times the strain containing plasmid pMG169 yielded higher batch productivities than the one containing pCQV2. The heat of fermentation was measured (using a continuous calorimetric method) as a means of partially implementing the temperature shift. The results differed from those obtained from widely used empirical correlations (which should not be used with recombinant strains). Design calculations were performed that allow batch and annual titres to be predicted under different fermenter configurations, scales, and modes of operation

A temperate river estuary is a sink for methanotrophs adapted to extremes of pH, temperature and salinity

River Tyne (UK) estuarine sediments harbour a genetically and functionally diverse community of methane-oxidizing bacteria (methanotrophs), the composition and activity of which were directly influenced by imposed environmental conditions (pH, salinity, temperature) that extended far beyond those found in situ. In aerobic sediment slurries methane oxidation rates were monitored together with the diversity of a functional gene marker for methanotrophs (pmoA). Under near in situ conditions (4-30°C, pH 6-8, 1-15gl-1 NaCl), communities were enriched by sequences affiliated with Methylobacter and Methylomonas spp. and specifically a Methylobacter psychrophilus-related species at 4-21°C. More extreme conditions, namely high temperatures ≥40°C, high ≥9 and low ≤5 pH, and high salinities ≥35gl-1 selected for putative thermophiles (Methylocaldum), acidophiles (Methylosoma) and haloalkaliphiles (Methylomicrobium). The presence of these extreme methanotrophs (unlikely to be part of the active community in situ) indicates passive dispersal from surrounding environments into the estuary

Swift Observations of GRB 050603: An afterglow with a steep late time decay slope

We report the results of Swift observations of the Gamma Ray Burst GRB 050603. With a V magnitude V=18.2 about 10 hours after the burst the optical afterglow was the brightest so far detected by Swift and one of the brightest optical afterglows ever seen. The Burst Alert Telescope (BAT) light curves show three fast-rise-exponential-decay spikes with $T_{90}$=12s and a fluence of 7.6$\times 10^{-6}$ ergs cm$^{-2}$ in the 15-150 keV band. With an $E_{\rm \gamma, iso} = 1.26 \times 10^{54}$ ergs it was also one of the most energetic bursts of all times. The Swift spacecraft began observing of the afterglow with the narrow-field instruments about 10 hours after the detection of the burst. The burst was bright enough to be detected by the Swift UV/Optical telescope (UVOT) for almost 3 days and by the X-ray Telescope (XRT) for a week after the burst. The X-ray light curve shows a rapidly fading afterglow with a decay index $\alpha$=1.76$^{+0.15}_{-0.07}$. The X-ray energy spectral index was $\beta_{\rm X}$=0.71\plm0.10 with the column density in agreement with the Galactic value. The spectral analysis does not show an obvious change in the X-ray spectral slope over time. The optical UVOT light curve decays with a slope of $\alpha$=1.8\plm0.2. The steepness and the similarity of the optical and X-ray decay rates suggest that the afterglow was observed after the jet break. We estimate a jet opening angle of about 1-2$^{\circ}$Comment: 14 pages, accepted for publication in Ap

Redshift Filtering by Swift Apparent X-ray Column Density

We remark on the utility of an observational relation between the absorption column density in excess of the Galactic absorption column density, $\Delta N_{\rm H} = N_{\rm H, fit} - N_{\rm H, gal}$, and redshift, z, determined from all 55 Swift-observed long bursts with spectroscopic redshifts as of 2006 December. The absorption column densities, $N_{\rm H, fit}$, are determined from powerlaw fits to the X-ray spectra with the absorption column density left as a free parameter. We find that higher excess absorption column densities with $\Delta N_{\rm H} > 2\times 10^{21}$ cm$^{-2}$ are only present in bursts with redshifts z$<$2. Low absorption column densities with $\Delta N_{\rm H} < 1\times 10^{21}$ cm$^{-2}$ appear preferentially in high-redshift bursts. Our interpretation is that this relation between redshift and excess column density is an observational effect resulting from the shift of the source rest-frame energy range below 1 keV out of the XRT observable energy range for high redshift bursts. We found a clear anti-correlation between $\Delta N_{\rm H}$ and z that can be used to estimate the range of the maximum redshift of an afterglow. A critical application of our finding is that rapid X-ray observations can be used to optimize the instrumentation used for ground-based optical/NIR follow-up observations. Ground-based spectroscopic redshift measurements of as many bursts as possible are crucial for GRB science.Comment: revised version including updates and the referee's comments, accepted for publication in the Astronomical Journal, 12 pages, 2 figures, 2 tables - v3 contains an update on the reference lis

GRB 060313: A New Paradigm for Short-Hard Bursts?

We report the simultaneous observations of the prompt emission in the gamma-ray and hard X-ray bands by the Swift-BAT and the KONUS-Wind instruments of the short-hard burst, GRB 060313. The observations reveal multiple peaks in both the gamma-ray and hard X-ray bands suggesting a highly variable outflow from the central explosion. We also describe the early-time observations of the X-ray and UV/Optical afterglows by the Swift XRT and UVOT instruments. The combination of the X-ray and UV/Optical observations provide the most comprehensive lightcurves to date of a short-hard burst at such an early epoch. The afterglows exhibit complex structure with different decay indices and flaring. This behavior can be explained by the combination of a structured jet, radiative loss of energy, and decreasing microphysics parameters occurring in a circum-burst medium with densities varying by a factor of approximately two on a length scale of 10^17 cm. These density variations are normally associated with the environment of a massive star and inhomogeneities in its windy medium. However, the mean density of the observed medium (n approximately 10^&#8722;4 cm^3) is much less than that expected for a massive star. Although the collapse of a massive star as the origin of GRB 060313 is unlikely, the merger of a compact binary also poses problems for explaining the behavior of this burst. Two possible suggestions for explaining this scenario are: some short bursts may arise from a mechanism that does not invoke the conventional compact binary model, or soft late-time central engine activity is producing UV/optical but no X-ray flaring.Comment: 28 pages, 6 figures. Accepted for publication in ApJ. Clarifications made and typos correcte

Governance, Coordination and Evaluation: the case for an epistemological focus and a return to C.E. Lindblom

While much political science research focuses on conceptualizing and analyzing various forms of governance, there remains a need to develop frameworks and criteria for governance evaluation (Torfing et al 2012). The post-positivist turn, influential in recent governance theory, emphasizes the complexity, uncertainty and the contested normative dimensions of policy analysis. Yet a central evaluative question still arises concerning the capacity of governance networks to facilitate ‘coordination’. The classic contributions of Charles Lindblom, although pre-dating the contemporary governance literature, can enable further elaboration of and engagement with this question. Lindblom’s conceptualisation of coordination challenges in the face of complexity shares with post-positivism a recognition of the inevitably contested nature of policy goals. Yet Lindblom suggests a closer focus on the complex, dynamically evolving, broadly ‘economic’ choices and trade-offs involved in defining and delivery policy for enabling these goals to be achieved and the significant epistemological challenges that they raise for policy-makers. This focus can complement and enrich both post-positivist scholarship and the process and incentives-orientated approaches which predominate in contemporary political science research on coordination in governance. This is briefly illustrated through a short case study evaluating governance for steering markets towards delivering low and zero carbon homes in England

Root-cause analyses of missed opportunities for the diagnosis of colorectal cancer in patients with inflammatory bowel disease

Background: Colonoscopic surveillance in patients with inflammatory bowel disease (IBD) leads to earlier detection of colorectal cancer (CRC) and reduces CRC-associated mortality. However, it is limited by poor adherence in practice. Aim: To identify missed opportunities to detect IBD-associated CRC at our hospital METHODS: We undertook root-cause analyses to identify patients with missed opportunities to diagnose IBD-associated CRC. We matched patients with IBD-associated CRC to patients with CRC in the general population to identify differences in staging at diagnosis and clinical outcomes. Results: Compared with the general population, patients with IBD were at increased risk of developing CRC (odds ratio 2.7 [95% CI 1.6-3.9], P < 0.001). The mean incidence of IBD-associated CRC between 1998 and 2019 was 165.4 (IQR 130.4-199.4) per 100 000 patients and has not changed over the last 20 years. Seventy-eight patients had IBD-associated CRC. Forty-two (54%) patients were eligible for CRC surveillance: 12% (5/42) and 10% (4/42) patients were diagnosed with CRC at an appropriately timed or overdue surveillance colonoscopy, respectively. Interval cancers occurred in 14% (6/42) of patients; 64% (27/42) of patients had a missed opportunity for colonoscopic surveillance where root-cause analyses demonstrated that 10/27 (37%) patients known to secondary care had not been offered surveillance. Four (15%) patients had a delayed diagnosis of CRC due to failure to account for previous colonoscopic findings. Seventeen (63%) patients were managed by primary care including seven patients discharged from secondary care without a surveillance plan. Matched case-control analysis did not show significant differences in cancer staging or 10-year survival outcomes. Conclusion: The incidence of IBD-associated CRC has remained static. Two-thirds of patients eligible for colonoscopic surveillance had missed opportunities to diagnose CRC. Surveillance programmes without comprehensive and fully integrated recall systems across primary and secondary care are set to fail.published version, accepted version (12 month embargo), submitted versionThis article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site